This invention relates to a series of bipolar lipids and to their use to deliver bioactive substances to cells.
To be effective, many pharmaceutical agents need to be efficiently delivered to the cytoplasm of a eucaryotic cell. For many low molecular weight compounds of low to moderate polarity this is not a problem since such molecules can pass directly through the plasma membrane of the cell and into the cytoplasm. Direct passage is not available to other compounds of greater polarity or high molecular weight and these generally enter the cell by receptor mediated endocytosis or phagocytosis. These mechanisms are not efficient however with all sizes and types of molecule. In particular, large, polyanionic compounds are not readily taken up by cells when delivered to them in aqueous solution.
One general solution to this problem is to couple any poorly transported pharmaceutical agent to a carrier which itself is readily taken up into the cytoplasm of a cell. This is not always satisfactory however, since coupling to the carrier may have an undesirable effect on the metabolism and/or antigenicity of the pharmaceutical agent and/or it may be difficult to recover the desired biological activity from the resulting conjugate once inside the cell.
An alternative solution is to formulate the pharmaceutical agent with a delivery vehicle which is soluble in aqueous solutions but which can also mimic naturally occurring cell membrane constituents. This encourages fusion of the vehicle with a cell membrane and subsequent delivery of any associated pharmaceutical agent to the cytoplasm.
Amphiphilic lipids have frequently been used for this purpose. These typically have a hydrophobic backbone composed of one or more hydrocarbons and a hydrophilic polar head group containing one or more ionisable groups, to facilitate the transport of macromolecules to and across the plasma membrane of cells and into the cytoplasm. The polarity of the head group may be controlled by the selection of the number and/or type of ionisable groups to achieve a range of negatively charged (anionic), neutral or positively charged (cationic) lipids.
For the delivery of polyanions it is generally advantageous to use cationic lipids. The advent of gene therapy and the need to deliver anionic molecules such as nucleic acids to mammalian cells has provided much impetus to the development of this class of lipids. First generation compounds include those with a monocation head group such as N-[1(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride [DOTMA; Felgner, P L and Ringold, G M, Nature, 337 387-388 (1989)], 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide [DMRIE; Zabner, J et al J. Biol. Chem, 270, 18997-19007 (1995)] and 3xcex2[N-(N1,N1-dimethylaminoethane)carbamoyl]cholesterol [DC-Chol; Farhood, H et al, Biochim. Biophys. Acta. 1111, 239-246 (1992)] and those with a polycation head group such as dioctadecylamidoglycylspermine [DOGS; Behr, J-P, et al, Proc. Natl. Acad. Sci. 86, 6982-6986 (1989)].
In an effort to improve the properties of these early compounds for in vivo delivery of polyanions many more cationic lipids have been developed in which the nature and size of the hydrophobic backbone and/or the cationic head group have been varied (see for example International Patent Specifications Nos. WO95/21931, WO96/10038, WO96/17823, WO96/18273, WO96/25508, WO96/26179, WO96/41606, WO97/18185, WO97/25339, WO97/3010 and WO97/31934).
The goal in the development of cationic lipids for in vivo use is to provide a molecule which is simple to use in a clinical setting; which is robust; which forms small stable complexes over wide pH and ionic strength ranges; which is non-toxic; and which is capable of delivering a high concentration of polyanion to a cell.
We have now developed a class of lipid which meets these requirements. Importantly, our lipids are capable of self-assembly and will form stable complexes in aqueous solutions. The lipids are able to efficiently compact polyanions to give defined particle sizes of less than 500 nm. The lipid-polyanion complex remains associated over wide pH and ionic strength ranges and is able to efficiently deliver high concentrations of polyanions to cells.
Thus according to one aspect of the invention we provide a bipolar lipid comprising a cationic head (1) a hydrophobic backbone (2) and a hydrophilic tail (3) in which:
(A) the cationic head comprises two or more cationic centres, each centre being covalently linked to one or more others by one or more carbon containing spacer groups;
(B) the hydrophobic backbone comprises one or more hydrocarbon chains; and
(C) the hydrophilic tail comprises one or more hydrophilic hydrocarbons each containing two or more atoms or groups capable of being solvated by water;
each of said components (1) to (3) being covalently linked head (1) to backbone (2) to tail (3) and arranged such that at least one hydrocarbon chain in the hydrophobic backbone (2) is covalently linked to a carbon atom of a spacer group in the cationic head (1) and each hydrophilic hydrocarbon in the hydrophilic tail (3) is covalently linked to a chain in the backbone (2) to achieve at least a ten atom spacing along the chain between the tail (3) and the head (1).
In the lipids according to the invention, each cationic centre in the cationic head (1) may be provided by one or more heteroatoms capable of retaining a positive charge at a pH in the range from around pH 2.0 to around pH 10.0. In practice, whether a heteroatom will retain a positive charge in this pH range will depend on the nature and number of any other atoms or groups attached to it. Thus particular examples of suitable cationic centres include primary, secondary, tertiary and quaternary amino groups, sulphonium and phosphonium groups.
The number of cationic centres may be varied as desired depending on the intended use of the lipid of the invention. At least two centres will be present, but three, four, five, six, seven, eight or more may be optionally incorporated. More than one type of centre may be present, for example mixtures of amino groups may be accommodated, and/or sulphonium and/or phosphonium groups.
In one general preference each cationic centre is an amino group. Particularly useful amino groups include primary and secondary amino groups. The number of cationic centres in the cationic head (1) will preferably be from three to six.
Each cationic centre will in general be separated from any other centre by spacer groups arranged to link the centres in a linear (straight and/or branched) or cyclic fashion. The overall effect may be a cationic head (1) which has a straight and/or branched linear structure, a cyclic structure, or a mixture of straight and/or branched linear and cyclic structures. More than one type of spacer group may be present in a cationic head (1). Where desired a spacer group may form a terminal group on the cationic head (1), acting as a substituent on a cationic centre rather than a group connecting centres together.
Each spacer group will in general be non-ionic and contain at least one carbon atom. Suitable groups include optionally substituted aliphatic, cycloaliphatic, heteroaliphatic, heterocycloaliphatic, aromatic or heteroaromatic groups.
Particular examples of optionally substituted aliphatic spacer groups include optionally substituted C1-10aliphatic chains such as optionally substituted straight or branched C1-6alkylene, C2-6alkenylene or C2-6alkynylene chains.
Heteroaliphatic spacer groups include the aliphatic chains just described but with each chain additionally containing one, two, three or four heteroatoms or heteroatom-containing groups. Particular heteroatoms or groups include atoms or groups L2 where L2 is as defined below for L1 when L1 is a linker atom or group. Each L2 atom or group may interrupt the aliphatic chain, or may be positioned at its terminal carbon atom to connect the chain to the atom or group R1.
Particular examples of aliphatic spacer groups include optionally substituted xe2x80x94CH2xe2x80x94, xe2x80x94CH2CH2xe2x80x94, xe2x80x94CH(CH3)xe2x80x94, xe2x80x94C(CH3)2xe2x80x94, xe2x80x94(CH2)2CH2xe2x80x94, xe2x80x94CH(CH3)CH2xe2x80x94, xe2x80x94(CH2)3CH2xe2x80x94, xe2x80x94CH(CH3)CH2CH2xe2x80x94, xe2x80x94CH2CH(CH3)CH2xe2x80x94, xe2x80x94C(CH3)2CH2xe2x80x94, xe2x80x94(CH2)4CH2xe2x80x94, xe2x80x94(CH2)5CH2xe2x80x94, xe2x80x94CHCHxe2x80x94, xe2x80x94CHCHCH2xe2x80x94, xe2x80x94CH2CHCHxe2x80x94, xe2x80x94CHCHCH2CH2xe2x80x94, xe2x80x94CH2CHCHCH2xe2x80x94, xe2x80x94(CH2)2CHCHxe2x80x94, xe2x80x94CCxe2x80x94, xe2x80x94CCCH2xe2x80x94, xe2x80x94CH2CCxe2x80x94, xe2x80x94CCCH2CH2xe2x80x94, xe2x80x94CH2CCCH2xe2x80x94, or xe2x80x94(CH2)2CCxe2x80x94 chains. Where appropriate each of said chains may be optionally interrupted by one or two atoms and/or groups L2 to form an optionally substituted heteroaliphatic spacer group. Particular examples include optionally substituted xe2x80x94L2CH2xe2x80x94, xe2x80x94CH2L2CH2xe2x80x94, xe2x80x94L2 (CH2)2xe2x80x94, xe2x80x94CH2L2(CH2)2xe2x80x94, xe2x80x94(CH2)2L2CH2xe2x80x94, xe2x80x94L2(CH2)3xe2x80x94 and CH2)2L2(CH2)2xe2x80x94 chains. The optional substituents which may be present on aliphatic or heteroaliphatic spacer groups include one, two, three or more substituents selected from halogen atoms, e.g. fluorine, chlorine, bromine or iodine atoms, or hydroxyl, C1-6alkoxy, e.g. methoxy or ethoxy, haloC1-6alkoxy, e.g. halomethoxy or haloethoxy such as difluoromethoxy or trifluoromethoxy, thiol, or C1-6alkylthio e.g. methylthio or ethylthio. Particular examples of substituted spacer groups include those specific chains just described substituted by one, two, or three halogen atoms such as fluorine atoms, for example chains of the type xe2x80x94CH(CF3)xe2x80x94, xe2x80x94C(CF3)2xe2x80x94 xe2x80x94CH2CH(CF3)xe2x80x94, xe2x80x94CH2C(CF3)2xe2x80x94, xe2x80x94CH(CF3)xe2x80x94 and xe2x80x94C(CF3)2CH2xe2x80x94.
Optionally substituted cycloaliphatic spacer groups in the cationic head (1) include optionally substituted C3-10 cycloaliphatic groups. Particular examples include optionally substituted C3-10cycloalkylene, e.g. C3-7cycloalkylene, C3-10cycloalkenylene e.g. C3-7cycloalkenylene or C3-10cycloalkynylene e.g. C3-7cycloalkynylene groups.
Particular examples of cycloaliphatic spacer groups include optionally substituted cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, 2-cyclobuten-1-ylene, 2-cyclopenten-1-ylene and 3-cyclopenten-1-ylene groups.
Optionally substituted heterocycloaliphatic spacer groups include the optionally substituted cycloaliphatic groups just described but with each group additionally containing one, two, three or four heteroatoms or heteroatom-containing groups L2 as just defined.
The optional substituents which may be present on the cycloaliphatic or heterocycloaliphatic spacer groups include one, two, three or more substituents selected from halogen atoms C1-6alkyl, e.g. methyl or ethyl, haloC1-6alkyl, e.g. halomethyl or haloethyl such as difluoromethyl or trifluoromethyl, hydroxyl, C1-6alkoxy, e.g. methoxy or ethoxy, haloC1-6alkoxy, e.g. halomethoxy or haloethoxy such as difluoromethoxy or trifluoromethoxy, thiol, or C1-6alkylthio e.g. methylthio or ethylthio groups.
Optionally substituted aromatic spacer groups include for example monocyclic C6-12 aromatic groups, such as optionally substituted phenylene.
Optionally substituted heteroaromatic spacer groups, include for example optionally substituted monocyclic C1-9 heteroaromatic groups containing for example one, two, three or four heteroatoms selected from oxygen, sulphur or nitrogen atoms. Monocyclic heteroaromatic groups include for example five- or six-membered heteroaromatic groups containing one, two, three or four heteroatoms selected from oxygen, sulphur or nitrogen atoms.
Optional substituents which may be present on the aromatic or heteroaromatic spacer groups include one, two, three or more substituents selected from those just described in relation to cycloaliphatic and heterocycloaliphatic spacer groups.
In one general preference each spacer group in the cationic head (1) is preferably an optionally substituted straight or branched C1-6alkylene chain. Particularly useful chains include xe2x80x94(CH2)2xe2x80x94, xe2x80x94(CH2)3xe2x80x94 and xe2x80x94CH2)4xe2x80x94 chains.
In the lipids of the invention at least one spacer group connecting two cationic centres is covalently linked through one of its carbon atoms to a hydrocarbon chain of the hydrophobic backbone (2). Where desired any other available carbon atom or heteroatom in the, or any other, spacer group, or any available atom in a cationic centre, may be additionally linked to the same or other hydrocarbon chains making up the backbone (2). It is generally preferred however to link the backbone (2) and cationic head (1) at one carbon atom in one spacer group.
The hydrophobic backbone (2) in the lipids according to the invention may comprise one or more hydrocarbon chains. Each hydrocarbon may be for example an optionally substituted straight or branched aliphatic or heteroaliphatic chain containing a minimum of ten up to a maximum of around one hundred chain-linked atoms as described in more detail below. The hydrocarbon may be attached either directly or indirectly through a linker atom or group to the cationic head (1). Particular examples of suitable linker groups are those represented by the group L1 described below. As explained above, more than one hydrocarbon chain may be attached to the head group but a preferred class of lipids according to the invention has one or two hydrocarbon chains as just described indirectly linked through a linker atom or group to a carbon atom in a spacer group connecting two cationic centres in the cationic head (1).
The hydrophilic tail (3) in the lipids according to the invention may in general be one or more hydrophilic hydrocarbons having little or no overall positive or negative charge and containing a minimum of two up to a maximum of around one hundred atoms or groups capable of being solvated by water. Each hydrophilic hydrocarbon in the hydrophilic tail (3) may be attached directly or indirectly through a linker atom or group to a hydrocarbon chain of the hydrocarbon backbone (2). The attachment point may be anywhere on the hydrocarbon chain provided that it is at least ten atoms along the chain, excluding branches, from the terminal carbon atom connecting the hydrophobic backbone (2) to the cationic head (1). In one general preference the attachment point may be at a terminal carbon atom of a hydrocarbon chain distal to the chain carbon atom attached to the cationic head (1). Particular examples of suitable hydrophilic hydrocarbons which constitute the hydrophilic tail (3) are described in more detail below.
A particularly useful group of lipids according to the invention may be represented by the formula (1):
[R1]mxe2x80x94(L1)nxe2x80x94[xe2x80x94C(R2)(R3)(R4)]xe2x80x83xe2x80x83(1)
wherein R1 is a hydrocarbon chain optionally substituted by one or more hydrophilic hydrocarbons each containing two or more atoms or groups capable of being solvated by water, provided that at least one hydrocarbon chain is substituted by at least one hydrophilic hydrocarbon and each hydrophilic hydrocarbon is attached to the hydrocarbon chain to achieve at least a ten atom spacing along the chain between the hydrophilic hydrocarbon and the group xe2x80x94(L1)nxe2x80x94[xe2x80x94C(R2)(R3)(R4)];
m is an integer from 1 to 6;
L1 is a linker atom or group;
n is zero or the integer 1;
xe2x80x94[xe2x80x94C(R2)(R3)(R4)] is a cationic head in which R2 is a hydrogen atom or an optionally substituted aliphatic, cycloaliphatic, heteroaliphatic, heterocycloaliphatic, aromatic or heteroaromatic group optionally containing one or more cationic centres, and R3 and R4 which may be the same or different is each an optionally substituted aliphatic, cycloaliphatic, heteroaliphatic, heterocycloaliphatic, aromatic or heteroaromatic group containing one or more cationic centres, or R3 and R4 together with the carbon atom to which they are attached form a cycloaliphatic, heterocycloaliphatic, aromatic or heteroaromatic group containing two or more cationic centres;
and the salts, solvates and hydrates thereof.
In the compounds of formula (1), the optionally substituted aliphatic, cycloaliphatic, heteroaliphatic, heterocycloaliphatic, aromatic or heteroaromatic group represented by R2, R3 and R4 may each be an optionally substituted C1-30 aliphatic, C3-10 cycloaliphatic, C1-30 heteroaliphatic, C3-10 heterocycloaliphatic, C6-12 aromatic or C1-9 heteroaromatic group, each containing one or more cationic centres. Particular examples of such groups include those generally and specifically described above in relation to the spacer groups present in the cationic head (1) with the additional presence of one or more cationic centres as defined herein.
In general in the lipids of the invention when the hydrophobic backbone (2) and cationic head (1) are joined indirectly by a linker atom or group, as represented by L1 in compounds of formula (1) when n is 1, then the linker atom or group-may be any multivalent atom or group. Particular examples of suitable linker atoms or groups include those of formula xe2x80x94(Alk1)r(X1)s(Alk2)txe2x80x94 where X1 is an xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94 atom or a xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94C(S)xe2x80x94, xe2x80x94S(O), xe2x80x94S(O)2xe2x80x94 xe2x80x94N(R5)xe2x80x94, [where R5 is a hydrogen atom, straight or branched alkyl group such as a methyl or ethyl group or an xe2x80x94Alk1X1xe2x80x94 chain], xe2x80x94CON(R5)xe2x80x94, xe2x80x94OC(O)N(R5)xe2x80x94, xe2x80x94CSN(R5)xe2x80x94, xe2x80x94N(R5)COxe2x80x94, N(R5)C(O)Oxe2x80x94, xe2x80x94N(R5)CSxe2x80x94, xe2x80x94S(O)N(R5)xe2x80x94, xe2x80x94S(O)2N(R5)xe2x80x94, xe2x80x94N(R5)S(O)xe2x80x94, xe2x80x94N(R5)S(O)2xe2x80x94, xe2x80x94N(R5)CON(R5)xe2x80x94, or xe2x80x94N(R5)SO2N(R5)xe2x80x94 group [where any of these groups contains two R5 substituents these may be the same or different]; Alk1 and Alk2 which may be the same or different is each an optionally substituted straight or branched C1-6alkylene, C2-6alkenylene or C2-6alkynylene chain optionally interrupted or terminated by one or more, e.g. one, two or three, carbocyclic or heterocarbocyclic groups and/or heteroatoms or heteroatom containing groups X1 as just defined, and r, s, and t, which may be the same or different, is each zero or the integer 1, provided that when one of r, s or t is zero at least one of the remainder is the integer 1.
Carbocyclic groups which may interrupt the groups Alk1 and Alk2 include for example optionally substituted C4-8cycloalkyl, e.g. optionally substituted cyclopentyl or cyclohexyl groups, or optionally substituted C4-8cycloalkenyl, e.g. optionally substituted cyclopentenyl or cyclohexenyl groups. Heterocarbocyclic groups include for example carbocyclic groups of the types just mentioned containing one or more heteroatoms or heteroatom-containing groups X1 as defined above. Optional substituents which may be present on the chains represented by Alk1 and Alk2 and the carbocyclic or heterocarbocyclic groups which can interrupt or terminate them include one, two or three substituents selected from halogen atoms, e.g. fluorine, chlorine, bromine or iodine atoms or C1-3alkyl, e.g. methyl or ethyl, or C1-3alkoxy e.g. methoxy or ethoxy groups.
It will be appreciated that the linker atom or group will be at least divalent in the instance where one hydrocarbon chain in the hydrophobic backbone (2) is attached to it. Where it is desired to attach more than one hydrocarbon chain to the linker the latter will need to be selected with an appropriate valency and this will generally mean that at least one of Alk1 or Alk2 will need to be present in a branched form and with the requisite number of X1 atoms or groups to achieve the desired coupling.
Particular examples of linker groups include groups of formula xe2x80x94X1Alk2xe2x80x94 where X1 is as defined above and Alk2 is an optionally substituted xe2x80x94CH2xe2x80x94, xe2x80x94(CH2)2xe2x80x94, xe2x80x94(CH2)3xe2x80x94, xe2x80x94(CH2)4xe2x80x94, xe2x80x94(CH2)5xe2x80x94 or xe2x80x94(CH2)6xe2x80x94 chain; groups of formula [X1]2Alk1X1Alk2 where Alk1 is a xe2x80x94CH2CH less than  group and X1 and Alk2 are as just defined or a group of formula [X1]2Alk1Alk2 where X1, Alk1 and Alk2 are as just defined.
Each hydrocarbon chain in the hydrophobic backbone (2) of the lipids according to the invention and as represented by R1 in compounds of formula (1) may be a C10 up to about a C60 hydrocarbon chain, for example a C16 to C60 hydrocarbon chain such as a C18 to C48 hydrocarbon chain.
In particular, the chain may be an optionally substituted C10-60 aliphatic chain such as an optionally substituted straight or branched C10-60alkylene, C10-60alkenylene or C10-60alkynylene chain. Optional substituents which may be present on such chains include one or more halogen atoms, e.g. fluorine, chlorine, bromine or iodine atoms, or haloC1-6alkyl, e.g. xe2x80x94CF3 groups. Where desired each alkylene, alkenylene or alkynylene chain may be interrupted by one or more oxygen or sulphur atoms or optionally substituted C5-7cycloalkyl, e.g. cyclopentyl or cyclohexyl, C5-7cycloalkenyl, e.g. cyclopentenyl or cyclohexenyl, xe2x80x94C(O)xe2x80x94, xe2x80x94C(S)xe2x80x94, xe2x80x94C(O)N(R5)xe2x80x94, xe2x80x94C(S)N(R5)xe2x80x94, xe2x80x94N(R5)C(O)xe2x80x94, xe2x80x94N(R5)C(S)xe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94C(O)Sxe2x80x94, xe2x80x94OC(O)N(R5)xe2x80x94, xe2x80x94S(O)xe2x80x94, xe2x80x94S(O2)xe2x80x94, xe2x80x94S(O)N(R5)xe2x80x94, xe2x80x94S(O)2N(R5)xe2x80x94, xe2x80x94N(R5)S(O)xe2x80x94, xe2x80x94N(R5)S(O)2xe2x80x94, xe2x80x94N(R5)C(O)N(R5)xe2x80x94, xe2x80x94N(R5)C(S)N(R5)xe2x80x94, xe2x80x94N(R5)S(O)N(R5)xe2x80x94 or xe2x80x94N(R5)S(O)2N(R5)xe2x80x94 groups. Optional substituents which may be present on cycloalkyl or cycloalkenyl groups of this type include one or more halogen atoms or haloalkyl groups as just described. It will be appreciated that when the hydrocarbon chain in the hydrophobic backbone (2) is an alkenylene or alkynylene chain it may have more than one unsaturated group.
As generally explained above, the hydrophilic tail (3) in the lipids according to the invention may be formed by one or more hydrophilic hydrocarbons, each attached to a hydrocarbon chain in the hydrophobic backbone (2), for example as generally represented by R1 in compounds of formula (1). Each hydrophilic hydrocarbon may be an aliphatic, heteroaliphatic, cycloaliphatic, polycycloaliphatic, heterocycloaliphatic or polyheterocycloaliphatic group. Particular examples of aliphatic groups include alkyl, alkenyl or alkynyl groups. Cycloaliphatic groups include cycloalkyl or cycloalkenyl groups. Polycycloaliphatic groups include two or more cycloalkyl or cycloalkenyl groups either joined directly or indirectly through a linker atom or group, for example a linker atom or group L2 where L2 is an atom or group as described above for the group L1. Each of these aliphatic, cycloaliphatic or polycycloaliphatic groups may be optionally interrupted by one or more heteroatoms or heteroatom-containing groups, for example of the type described above in relation to the group L1 to yield heteroaliphatic, heterocycloaliphatic or polyheterocycloaliphatic hydrocarbon groups. In general, each hydrophilic hydrocarbon group forming the hydrophilic tail (3) may contain from one carbon atom to around two hundred carbon atoms.
Each hydrophilic hydrocarbon contains two or more atoms or groups capable of being solvated by water. Examples of such groups include oxygen atoms (xe2x80x94Oxe2x80x94) or oxygen-containing groups. Oxygen atoms may form part of a heteroaliphatic, heterocycloaliphatic or polycycloheteroaliphatic group as just described. Oxygen-containing groups may be substituents present on the various hydrocarbons just mentioned and include for example hydroxyl, amide and alkoxy groups such as methoxy or ethoxy groups. In general the number of groups capable of being solvated by water in each hydrocarbon will range from two to around two hundred.
Particular examples of suitable hydrophilic hydrocarbons include polyols. Suitable polyols include naturally occurring polyols such as sugars and derivatives thereof, and synthetic polyols. Particular sugars include mono- and oligosaccharides. Sugar derivatives include glycosides in which a non-ionic aliphatic or heteroaliphatic group (for example of the type described herein) is joined to a sugar by a glycosidic linkage. Monosaccharides include for example open-chain or cyclic compounds containing three to eight, e.g. five or six, carbon atoms and at least two hydroxyl substituents. Oligosaccharides include for example at least two monosaccharides as just defined linked together by a glycosidic linkage. More than one type of monosaccharide may be present to yield a homo- or heterooligosaccharide.
Alternatively the hydrophilic hydrocarbon may be a polyether, for example a poly(alkylene oxide) and derivatives thereof, such as poly(ethylene oxide), poly(propylene oxide) or methoxy poly(ethylene oxide), a poly(oxyalkylated alcohol) or a poly(alkenylene alcohol) or poly(alkynylene alcohol) such as poly(vinyl alcohol). The hydrocarbons may in general be straight or branched. Where desired co-polymers of these hydrocarbons may be used.
Each hydrophilic hydrocarbon may be linked directly or indirectly to a hydrocarbon chain in the hydrophobic backbone (2). For indirect linkage a linker atom or group may be employed, for example an atom or group L3 where L3 is as defined above as for the linker atom or group L1. Where the group L3 is multivalent, for example when it is a branched alkylene chain containing more than one X1 atom or group, more than one hydrophilic hydrocarbon may be attached to it.
A particularly useful group of compounds according to the invention has the formula (1a):
[R7]pxe2x80x94(L3)qxe2x80x94[R6]mxe2x80x94(L1)nxe2x80x94[xe2x80x94C(R2)(R3)(R4)]xe2x80x83xe2x80x83(1a)
wherein
R2, R3, R4, L1, m and n are as defined for formula (1);
R6 is a hydrocarbon chain;
L3 is a linker atom or group;
R7 is a hydrophilic hydrocarbon containing two or more atoms or groups capable of being solvated by water;
q is zero or an integer from one to six;
p is an integer from one to six;
and the salts, solvates and hydrates thereof, provided that each R7 or L3 group, when present, is attached to a group R6 to achieve at least a ten atom spacing along R6 between R7 or L3 and the group xe2x80x94(L1)nxe2x80x94[C(R2)(R3)(R4)].
In the compounds of formula (1a) the hydrocarbon chain represented by R6 may be a C10 up to about a C60 hydrocarbon chain as generally and more particularly described above in relation to the group R1. The hydrophilic hydrocarbon R7 may similarly be a hydrophilic hydrocarbon as described previously in relation to the group R1. The group L3 may be a linker atom or group as just defined.
The cationic head (1) in the lipids according to the invention will preferably be a group xe2x80x94C(R2)(R3)(R4) as described above in relation to the compounds of formulae (1) and (1a). In groups of this type, R2 is preferably a hydrogen atom, and R3 and R4 is each preferably a group xe2x80x94Sp1[WSp2]bWSp3 or xe2x80x94Sp1[WSp2]bWH in which Sp1, Sp2 and Sp3, which may be the same or different, is each a spacer group as defined above, W is a cationic centre as defined herein and b is zero or an integer from one to six.
In particular groups of this type, the cationic centre W is preferably a xe2x80x94NHxe2x80x94 group. Sp1, Sp2 and Sp3, which may be the same or different, is each preferably an optionally substituted C1-6alkylene chain. b is preferably an integer from one to three.
Particularly useful cationic heads (1) in compounds of the invention include those of formula xe2x80x94CH[Sp1NHSp2NH2]2, xe2x80x94CH[Sp1NHSp2NHSp2NH2]2 or xe2x80x94CH[Sp1NHSp2NHSp2NHCH3]2 where each Sp1 and Sp2 group is the same or different and is an optionally substituted C1-6alkylene chain, particularly wherein Sp1 is xe2x80x94CH2xe2x80x94 and each Sp2 is xe2x80x94(CH2)3xe2x80x94 or xe2x80x94(CH2)4xe2x80x94.
In general in the lipids according to the invention the hydrophobic backbone (2) preferably comprises two or, especially one hydrocarbon chain as defined herein. Thus m in formulae (1) and (1a) is preferably an integer 2 or, especially, an integer 1. Each hydrocarbon chain, for example as represented by R1 and R6 in formulae (1) and (1a) respectively, is preferably linear and in particular is a linear, optionally substituted C16-38alkylene chain. Optionally substituted C18-24alkylene chains are particularly useful.
In general each hydrocarbon chain in the hydrophobic backbone (2) is preferably linked indirectly to the cationic head (1) through a linker atom or group. The linker atom or group may be for example an atom or group L1 as defined herein and thus in the compounds of formulae (1) and (1a) for example n is preferably the integer 1.
Preferred linkers include those of formula xe2x80x94X1Alk2xe2x80x94 or xe2x80x94[X1]2Alk1X1Alk2xe2x80x94 where X1, Alk1 and Alk2 are as defined previously. Particularly useful linkers of these types are those wherein Alk2 is a xe2x80x94(CH2)4xe2x80x94, xe2x80x94(CH2)5xe2x80x94 or, especially, xe2x80x94(CH2)6xe2x80x94 chain. X1in these linkers is preferably a xe2x80x94CONHxe2x80x94 group. Alk1 when present is preferably a xe2x80x94CH2xe2x80x94CH less than  chain.
In another general preference each hydrocarbon chain in the hydrocarbon backbone (2) has two, or especially one, hydrophilic hydrocarbon attached to it. Each hydrophilic hydrocarbon is preferably attached to the terminal carbon atom of the hydrocarbon chain distal to the chain carbon atom attached to the cationic head (1). Preferably the hydrophilic hydrocarbon and hydrocarbon chain are indirectly linked through a linker atom or group. Thus in one particular preference in compounds of formula (1a) q is the integer 1 and p is the integer 1 or 2.
In compounds of this type and in general the group L3 may preferably be an atom or group xe2x80x94X1xe2x80x94, xe2x80x94X1Alk1X1xe2x80x94 or [X1Alk1]2X1Alk2X1xe2x80x94. Particularly useful L3 groups include xe2x80x94NHCOxe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94CONH(CH2)2NHCOxe2x80x94, or xe2x80x94[CONH(CH2)2xe2x80x94]2NCO(CH2)2CONHxe2x80x94 groups.
In general, the hydrophilic hydrocarbon, for example as represented by R7 in formula (1a) is preferably a synthetic polyol, a naturally occurring polyol such as mono- or disaccharide, or a poly(alkylene oxide) as defined herein. In particular R7 may be a poly(alkylene oxide) or a derivative thereof, especially a poly(ethylene oxide).
Particularly useful lipids according to the invention are those described in the Examples hereinafter, especially in Sections H and I.
The lipids according to the invention may generally be prepared by coupling appropriately functionalised cationic heads (1), hydrophobic hydrocarbons (2) and hydrophilic hydrocarbons (3) in a predetermined order. Standard chemical coupling techniques may be employed utilising starting materials containing one or more reactive functional groups such as acids, thioacids, anhydrides, acid halides, esters, imides, aldehydes, ketones and amines. Illustrative reactions are described in detail in the Examples hereinafter for the preparation of a number of lipids according to the invention and these may be readily adapted using different starting materials to provide other compounds of the invention.
Thus in one general approach a homo- or heterobifunctional hydrocarbon chain may first be coupled to a hydrophilic hydrocarbon or cationic head and the resulting product coupled as necessary to the remaining component to provide the lipid of the invention.
The homo- or heterobifunctional hydrocarbon chain may be any hydrocarbon chain described herein containing two different reactive functional groups of the types just described. Particularly useful groups include acids and thioacids and reactive derivatives thereof, and amines. These can be used to participate in acylation or thioacylation reactions to couple the hydrocarbon chain to an amine or acid as appropriate in any suitable hydrophilic hydrocarbon and/or cationic head.
Acylation or thioacylation may be achieved using standard conditions for reactions of this type. Thus, for example the reaction may be carried out in a solvent, for example an inert organic solvent such as an amide, e.g. a substituted amide such as dimethylformamide, an ether, e.g. a cyclic ether such as tetrahydrofuran, or a halogenated hydrocarbon, such as dichloromethane, at a temperature from around ambient temperature to the reflux temperature, optionally in the presence of a base such as an amine, e.g. triethylamine, or a cyclic amine, such as 1,8-diazabicyclo[5.4.0] undec-7-ene pyridine, dimethylaminopyridine, or N-methylmorpholine.
Where an acid is used the acylation may additionally be performed in the presence of a condensing agent, for example a diimide such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or N,Nxe2x80x2-dicyclohexylcarbodiimide, advantageously in the presence of a catalyst such as a N-hydroxy compound e.g. a N-hydroxytriazole such as 1-hydroxybenzotriazole or a N-hydroxyimide such as N-hydroxysuccinimide. Alternatively, the acid may be reacted with a chloroformate, for example ethylchloroformate, prior to reaction with the amine.
In the heterobifunctional hydrocarbon chain one of the reactive functional groups may need to be in a protected form prior to any coupling reaction to avoid its unwanted participation in the reaction. Similarly other functional groups when present in the hydrocarbon chain, or the intermediates used to generate the hydrophilic hydrocarbon and/or the cationic head may need to be in a protected form before these reagents can be used. Conventional protecting groups may be used in accordance with standard practice [see, for example, Green, T. W. in xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, John Wiley and Sons, 1991 and the Examples hereinafter].
Suitable heterobifunctional hydrocarbon chains are either known, readily available materials or may be obtained by synthesis using conventional techniques for example as described in the Examples hereinafter. Thus generally a heterobifunctional hydrocarbon chain of any desired length may be synthesised in one or more reactions using appropriately functionalised shorter chains. Thus in one example a shorter chain aldehyde may be reacted with a shorter chain phosphonium salt to yield a longer chain olefin of the desired length. In this particular example the reaction may be carried out in the presence of a base, for example an organometallic base such as an organolithium compound, a hydride such as sodium or potassium hydride or an alkoxide such as a sodium alkoxide e.g. sodium methoxide. The reaction may be performed in a suitable solvent, for example a polar aprotic solvent such as an alkyl sulphoxide, e.g. dimethylsulphoxide at a low temperature, for example around 0xc2x0 C. The starting aldehyde and phosphonium salt may be obtained from known starting alcohols and halides respectively using conventional procedures. Where desired, the olefin obtained above may be hydrogenated using hydrogen and a catalyst, for example Pearlman""s catalyst, to yield the corresponding saturated hydrocarbon chain.
Where it is desired to obtain hydrocarbon chains containing one or more heteroatoms or heteroatom-containing groups these may be synthesised from smaller chains containing functional groups which can be chemically coupled, for example by acylation or thioacylation as generally described above.
Suitable functionalised hydrophilic hydrocarbons or cationic heads for coupling to the heterobifunctional hydrocarbon chain are either readily available or may be synthesised from known materials by conventional methods for example as described in the Examples hereinafter.
The advantageous properties of the lipids according to the invention may be demonstrated using the small scale tests described hereinafter in the Examples. In these the lipids can be shown to efficiently compact any bioactive substance, and to self-assemble with the substance in aqueous solution to yield stable complexes which remain associated over wide pH and ionic strength ranges and which will efficiently deliver the substance to eucaryotic cells.
The lipids can thus be expected to be of use for the delivery of bioactive substances to cells, particularly eucaryotic cells, in vitro and especially in vivo. Particular general uses to which the lipids may be put thus include for the delivery of bioactive substances to cells in culture, and in human medicine for the delivery of therapeutic or diagnostic agents, or agents which can generate a host immune response for vaccine or other immuno-modulatory purposes. The lipids are particularly well suited for delivering bioactive polyanions, especially nucleic acids, and are of particular use to modify a host""s genotype or its expression.
Thus, in another aspect of the invention we provide a lipid complex characterised in that it comprises a bipolar lipid comprising a cationic head (1) a hydrophobic backbone (2) and a hydrophilic tail (3) in which:
(A) the cationic head comprises two or more cationic centres, each centre being covalently linked to one or more others by one or more carbon containing spacer groups;
(B) the hydrophobic backbone comprises one or more hydrocarbon chains; and
(C) the hydrophilic tail comprises one or more hydrophilic hydrocarbons each containing two or more atoms or groups capable of being solvated by water;
each of said components (1) to (3) being covalently linked head (1) to backbone (2) to tail (3) and arranged such that at least one hydrocarbon chain in the hydrophobic backbone (2) is covalently linked to a carbon atom of a spacer group in the cationic head (1) and each hydrophilic hydrocarbon in the hydrophilic tail (3) is covalently linked to a chain in the backbone (2) to achieve at least a carbon atom spacing along the chain between the tail (3) and the head (1), in association with one or more bioactive substances.
In the complexes according to the invention, each bioactive substance may be for example a pharmacologically active agent, including an endosomolytic agent, a diagnostic agent or any agent able to modify the genotype and/or phenotype of a cell.
Particular examples of such substances include bioactive proteins, peptides, polysaccharides, nucleic acids including synthetic polynucleotides, oligonucleotides and derivatives thereof, lipids, glycolipids, lipoproteins, lipopolysaccharides and viral, bacterial, protozoal, cellular or tissue fractions.
Where desired the complexes according to the invention may contain two or more different bipolar lipids of the invention and such lipid mixtures form a further particular aspect of the invention. Especially useful mixtures include those containing two or more bipolar lipids of the invention which differ from each other in the nature of the hydrophilic tail present in each. The proportion of each lipid in complexes of this type may be manipulated to obtain complexes with different physio-chemical properties, for example overall surface charge and/or particle size, tailored to meet the intended use of the complex. Thus for example in one advantageous lipid complex containing two or more bipolar lipids, one of the lipids has a hydrophilic tail formed by a poly(alkyene oxide) or a derivative thereof as defined herein, while each of the others has a hydrophilic tail formed by a synthetic or naturally occurring polyol as described previously. The proportion of the first poly(alkylene oxide)-containing lipid may be varied in such complexes so that the mole ratio of first lipid to second and other lipids is from 1:10000 to 1:1, advantageously from around 1:1000 to around 1:20, especially around 1:10. Complexes of these types, particularly where the poly(alkylene oxide) is poly(ethylene oxide), may be obtained which advantageously have zero surface charge and do not aggregate when left in solution and which additionally are able to compact a bioactive substance to give small particles of 150 nm and below, particularly 100 nm and below, especially around 80-85 nm.
The lipids according to the invention are particularly suited for delivering polyanions to cells and preferred lipid complexes of the invention thus include lipid-polyanion complexes in which the polyanion may be any of the above-mentioned bioactive substances possessing a net negative charge. Particular polyanions include-nucleic acids, for example single or double stranded, circular or supercoiled DNA or RNA and derivatives thereof. Where desired the DNA may be part of a structure such as a plasmid.
The lipid complexes will in general comprise a lipid according to the invention and a bioactive substance in a weight ratio of around 0.1:1 to around 100:1, for example around 1:1 to around 50:1. The complexes may be formed as liquids, by initially mixing one or more bipolar lipids according to the invention, and a bioactive substance together advantageously in an aqueous solvent using conventional procedures. Where desired the solvent may be removed, for example by lyophilisation, to obtain a solid lipid complex.
The lipid complexes according to the invention may be formulated with other materials such as one one or more other lipids or other pharmaceutically acceptable carriers, excipients or diluents and the invention extends to such compositions. In this aspect of the invention the xe2x80x9cotherxe2x80x9d lipid may be for example selected from any known neutral and/or cationic lipid, for example selected from those described herein in the introduction to the invention (see page 2) and also especially including DOPE and other cholesterol derivatives such as cholesterol hemisuccinate. Particularly useful formulations of this type are those wherein the bipolar lipid of the invention has a poly(alkylene oxide) tail, especially a poly(ethylene oxide) tail.
Particular compositions include liposome formulations, prepared using conventional liposome technology. Otherwise, the compositions may take any other supermolecular form suitable for oral, buccal, parenteral, nasal, topical or rectal administration, or a form suitable for administration by inhalation or insufflation.
For oral administration, the compositions may take the form of, for example, tablets, lozenges or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium glycollate); or wetting agents (e.g. sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents, emulsifying agents, non-aqueous vehicles and preservatives. The preparations may also contain buffer salts, flavouring, colouring and sweetening agents as appropriate.
Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.
The complexes of the invention may be formulated for parenteral administration by injection, including bolus injection or infusion or particle mediated injection. Formulations for injection may be presented in unit dosage form, e.g. in glass ampoule or multi dose containers, e.g. glass vials or a device containing a compressed gas such as helium for particle mediated administration. The compositions for bolus injection or infusion may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising, preserving and/or dispersing agents. Alternatively, the complex may be in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use. For particle mediated administration the complex may be coated on particles such as microscopic gold particles.
In addition to the formulations described above, the complexes may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation or by intramuscular injection.
For nasal administration or administration by inhalation, the complexes may be conveniently delivered in the form of an aerosol spray presentation for pressurised packs or a nebuliser, with the use of suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas or mixture of gases.
The complexes may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack or dispensing device may be accompanied by instructions for administration.
The quantity of lipid complex required for any particular application will to a large extent depend on the nature of the bioactive substance being delivered. Another important factor will include whether the lipid complex is intended for in vitro or in vivo use. If the latter the route of administration and particular formulation chosen as well as factors such as the age and condition of the subject will govern the quantity of lipid complex used. In general however up to around 50 mg of lipid complex can be used for every kilogram of body weight.